Transcriptome sequencing reveals altered ciliogenesis under hypoxia in nasal epithelial cells from chronic rhinosinusitis with nasal polyps

Abstract Background Hypoxia is considered a key factor in the pathogenesis of chronic rhinosinusitis with nasal polyps (CRSwNP). However, the specific mechanism driving polypogenesis under hypoxic conditions is unclear. This study aimed to explore hypoxia‐induced alterations in the transcriptome of human nasal epithelial cells (HNECs) in vitro. Methods HNECs derived from the tissue of patients with CRSwNP were established as air–liquid interface (ALI) cultures. Confluent cultures were kept submerged or treated with cobalt chloride (CoCl2) to induce hypoxia. Transcriptome analysis was used to identify key mRNAs involved in this process. Real‐time PCR (RT–PCR), Western blotting, and immunofluorescence were used to observe the effects of hypoxia on ciliogenesis. Results Numerous genes, biological processes and pathways were altered under submerged culture conditions or after CoCl2 treatment. Analysis of the results under both hypoxic conditions revealed that the transcriptional program responsible for ciliogenesis was significantly impaired. Downregulation of cilia‐related genes and inhibition of ciliated cell differentiation under hypoxia were confirmed by RT–PCR, Western blot and immunofluorescence analyses. Conclusion Hypoxia impairs ciliogenesis and ciliary function in HNECs, which might play a role in the pathogenesis of CRSwNP.


| INTRODUCTION
Chronic rhinosinusitis (CRS) is a multifunctional inflammatory disease of the nasal cavity and sinus mucosa lasting more than 12 weeks, and it imposes a heavy burden not only on individual patients but also on the economy and society. 1,2 CRS with nasal polyps (CRSwNP) accounts for approximately 20% of CRS cases and has more severe clinical symptoms and a higher recurrence rate. 2 Hypoxia is defined as decreased availability of oxygen in tissues, and a hypoxic state in the airway epithelium has been reported to be associated with several chronic airway diseases, such as allergic rhinitis, asthma, and chronic obstructive pulmonary disease. 9 Moreover, hypoxia is considered to play an important role in the pathogenesis of CRS, and oxygen levels were found to be substantially decreased in the sinus cavities of patients with CRS. 10 The expression of hypoxia-inducible factor (HIF)-1α, an essential factor for oxygen homeostasis and a well-known hypoxia marker, has been reported to be upregulated in the sinus mucosal epithelium in patients with CRSwNP. 11 Recent study by Zhong et al. 12 demonstrated that HIF-1α induces the expression and activation of pyrin domain containing 3 (NLRP3) inflammasome by M1 macrophages in noneosinophilic CRSwNP, contributing to CRSwNP pathogenesis. Additional studies have reported that hypoxia can induce mucus hyperproduction, epithelial barrier disruption and tissue remodeling in the upper airway epithelium. 9 Despite these findings, the effects of hypoxia on human nasal epithelial cells (HNECs) and the potential underlying regulatory mechanism are incompletely elucidated.
The aim of the present study was to establish an in vitro model of hypoxia in HNECs and to investigate the alterations in epithelial cell function under hypoxia by transcriptome analysis to identify the key genes, biological processes (BPs) and pathways regulated during this process.

| Subjects
Ten subjects with CRSwNP were recruited for this study. Patients with CRSwNP were diagnosed according to the European Position Paper on Rhinosinusitis and Nasal Polyps 2020 (EPOS 2020) guidelines. 1 The diagnosis of allergic rhinitis was evaluated based on Allergic Rhinitis and its Impact on Asthma (ARIA) 2008 update. 13 The diagnosis of comorbid asthma was made according to the Global Initiative for Asthma 2019 guidelines. Subjects with severe uncontrolled systemic diseases, immunodeficiency, fungal sinusitis, and aspirin-exacerbated respiratory disease (AERD) were excluded. None of the patients had been treated with oral/nasal corticosteroids or antibiotics during the 4 weeks before surgery. Additionally, none of the patients had received biologics during the 6 months before surgery. The demographics and clinical characteristics of these patients are shown in Table S1. Nasal polyp tissues were isolated from patients with CRSwNP during surgery. The Ethics Committee of Beijing Tongren Hospital (TRECKY2019-050) approved this study, and all patients signed informed consent forms before enrollment.

| Cell culture and hypoxia model establishment
Primary HNECs were cultured as previously described. 14  containing 50 nM all-trans retinoic acid solely on the basolateral side.
The ALI culture medium in the basolateral compartment was replaced every other day.
The establishment of the hypoxia model was initiated 7 days after the establishment of the ALI culture. We used submerged culture and CoCl2 treatment to induce cellular hypoxia, as described in other publications. [15][16][17] Specifically, cell cultures from 5 CRSwNP patients were used to establish the submerged culture hypoxia model by exposing cells to 100 µL of ALI medium (added to the apical compartment for 4 h daily for 7 days), whereas the control cells were maintained at a ALI without treatment (ALI control, ALI). Cell cul-

| RNA sequencing (RNA-seq) and data analysis
For RNA-seq, total RNA extraction, library construction, sequencing and data analysis were carried out by Novogene (Beijing, China).

| Real-time polymerase chain reaction (RT-PCR)
After incubation, HNECs were lysed in RLT buffer, and total RNA was extracted using an RNeasy Kit (Qiagen, Hilden, Germany). Complementary DNA (cDNA) was synthesized using PrimeScriptTM RT Master Mix (TaKaRa Biotechnology). RT-PCR was performed using a SYBR Green assay (TaKaRa Biotechnology) according to the manufacturer's instructions. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the endogenous reference for mRNAs. Relative expression was calculated using the comparative cycle threshold method. The sequences of the primers used to amplify the target genes are presented in Table S2.

| Western blot analysis
After hypoxia treatment, HNECs were harvested by using RIPA lysis buffer containing 1% protease inhibitor for 30 min. The protein concentration was measured with a BCA kit (Beyotime, Shanghai, China). Then, cell lysates containing equal amounts of protein (20 µg) were separated by SDS-PAGE and transferred to PVDF membranes.

| Statistical analysis
GraphPad Prism Version 5.0 software (GraphPad Software, La Jolla, CA, USA) was used for the data analysis. All of the data are expressed as means � SEM, unless otherwise noted. The measurement data were normally distributed and were statistically analyzed by using a paired t test between the control and treatment groups. A two-tail value of p < 0.05 was considered to be statistically significant.

| Functional enrichment analysis of DE-mRNAs
The DE-mRNAs in the submerged culture and CoCl2-treated groups were subjected to GO term enrichment analysis, and the GO BP terms enriched with upregulated or downregulated DE-mRNAs are shown in Figure 2. Compared with the ALI control cells, the GO BP terms enriched with upregulated genes in the HNECs in the submerged culture were mainly associated with the hypoxia response (i.e., response to oxygen levels, response to decreased oxygen levels, and response to hypoxia), blood vessel morphogenesis, angiogenesis, the response to lipopolysaccharides, and the response to molecules of bacterial origin (Figure 2A Figure 2D). The upregulated gene-enriched BP terms common to both hypoxia treatments included response to hypoxia, blood vessel morphogenesis, angiogenesis, and female pregnancy, whereas the downregulated gene-enriched BP terms common to both hypoxia treatments were cilium organization, cilium assembly, microtubule cytoskeleton organization, chromosome segregation, sister chromatid segregation, and mitotic sister chromatid segregation.
A KEGG pathway enrichment analysis was performed to identify pathways enriched with DE-mRNAs in the submerged culture versus ALI control groups and in the CoCl2 versus untreated control groups.
As shown in Figure 3A

| Expression of cilia-related genes
The above RNA-seq data suggested alterations in the mRNA expression of cilia-related genes; therefore, we further analyzed the expression of several genes related to ciliogenesis and ciliary function under these two hypoxic conditions. Numerous genes

| Hypoxia inhibits ciliated cell differentiation
To determine whether submerged culture or CoCl2 treatment induces hypoxia in HNECs, HNECs were maintained in submerged culture for 4 h or treated with 100 µM CoCl2 for 24 h. Subsequently, the protein level of HIF-1α, which is a well-known hypoxia marker, as well as the mRNA expression of several hypoxia-response genes, were measured by using Western blotting and RT-PCR, respectively. Both the sub-   Notably, in addition to ciliogenesis-related processes, other BPs were also common to the two hypoxia treatment conditions. For example, blood vessel morphogenesis and angiogenesis were upregulated in HNECs under both submerged culture and CoCl2 treatment conditions. Many studies have indicated that abnormal angiogenesis and vascular permeability are important for the formation of nasal polyps. 25,26 Additionally, hypoxia-regulated angiogenesis has been reported to play an important role in various pathological conditions, such as solid tumors, vascular injury, and atherosclerotic lesion progression. 27 These findings indicate that hypoxia might contribute to NP propagation by leading to dysregulated angiogenesis. We will continue to focus on the roles of epithelium-derived angiogenic factors in the pathogenesis of nasal polyposis in follow-up research.
Furthermore, the results obtained from the two datasets were considerably different due to the different mechanisms of action in the two hypoxia models. The submerged culture creates a hypoxic environment for epithelial cell growth; therefore, we applied this cell culture system to gain insight into the impact of hypoxia on epithelial function in vivo. CoCl2 treatment of in vitro-cultured cells is a wellestablished method for inducing changes similar to those seen under hypoxia. 28 Our results showed that, in addition to enriching hypoxia-and cilia-related BP terms, the submerged culture also notably enriched the BP terms related to inflammation and immunity, such as responses to lipopolysaccharide and responses to molecules of bacterial origin. Additionally, this culture depleted BP terms, such as cell projection assembly, axoneme assembly, and microtubule-based movement. In addition, the upregulated DE-mRNAs after CoCl2 treatment were closely linked to metabolic process terms such as 4hydroxyproline metabolic process, carboxylic acid metabolic process, carbohydrate metabolic process, and pyruvate metabolic process,  30 Hypoxia was also demonstrated to be involved in the pathogenesis of CRSwNP through the regulation of Th17 immune responses of the nasal polyp epithelium. 31 These data indicate that hypoxia-induced dysregulation of the airway epithelial innate immune response may be associated with a compromised immunity and chronic inflammation of the airway, which highlight the essential work that is further needed to outline the role of tissue hypoxia in the regulation of immunity and inflammation in the pathophysiology of CRSwNP.
Our study had several limitations. First, we performed the experiments with only CRSwNP-derived epithelial cells, but we did not include healthy control cells, which may decrease the relevance of the findings. Further studies for comparing the difference in responses to hypoxia between inflamed and non-inflamed epithelial cells are necessary. Another limitation of this study was that the number of samples was small; thus, the sample sizes should be expanded for group comparisons of the effects of hypoxia on different phenotypes of CRSwNP. Thirdly, the molecular mechanisms underlying the influence of hypoxia on ciliary function remains to be elucidated. Lastly, in vitro experiments could not completely mimic in vivo conditions; therefore, these results need to be further verified in animal models in the future.
In summary, this study provides novel insight into the molecular basis of sinonasal ciliogenesis under hypoxic conditions, demonstrating that the gene transcription program responsible for ciliogenesis and ciliary function is significantly impaired under hypoxia, which might contribute to the disrupted mucociliary function in CRSwNP.